警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元
Summary
This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.
Abstract
There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal’s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.
Introduction
监测神经元活动中积极从事一个行为的任务的警报动物对理解神经系统的功能和组织的关键。从单个神经元单位电活动的细胞外记录一直是神经系统的主食工具，仍然是广泛使用在目前。各种电极类型和配置都可以根据具体实验的科学和技术要求。长期植入微型硬盘或电极阵列通常在自由活动的动物，包括鸟类，啮齿类动物，非人灵长类动物1-4使用。可替代地，通过外部显微急性穿透与金属或玻璃微电极通常用于从麻醉或头部限制动物来记录。玻璃微电极具有它们可以被用在juxtacellular或“细胞附着”的优势构明确地隔离单个神经元无事后秒杀分拣5的并发症的活性。这些电极从标题解剖的细胞或位置进一步允许记录，因为它们可以被用于注入的染料或神经解剖学示踪剂小存款，或甚至填充单个记录单元。这种配置已经在老鼠和鸟类6-10得到成功应用。目前所描述的技术，专注于juxtacellular监测和细胞外染料存款警报，头克制老鼠。注意juxtacellular罢了，这些染料存款不提供有关细胞形态和轴突的突起11的信息，不像单个细胞，但它们能精确解剖定位到约50微米，关键的是，有显著产量较高的警觉动物。对于单细胞juxtacellular填充仍然是作为用于解剖标签替代战略的信息。
简言之，在协议包括三个主要阶段。在第一阶段，将大鼠驯化身体约束在一个布袜子（ 图1）历时6天。在第二阶段中，头枕装置（ 图2）和记录室被手术植入，使得老鼠可以在多个后续的记录会话（ 图3）被保持在立体平面上;这个程序可以使实验者基于标准的参考坐标12为目标的大脑的特定子皮层区域的电生理研究。第三阶段涉及将大鼠在适当的夹具用于进行行为和电生理实验（ 图4），从一个石英毛细管构成电极（ 图5），使得juxtacellular神经元的录音是明确分离单个单元6-9，和标志着解剖LOCATI在记录现场与芝加哥天蓝染料（ 图6和7）组成。在录音同声行为进行监督;然而，该行为的技术细节将取决于每个实验的科学目标，因而超出了单个协议的范围。的实验程序，它可以被重复多天完成后，将动物安乐死。大脑是根据使用或者明场或荧光显微镜标准神经解剖技术开采和加工。
Protocol
Representative Results
Discussion
试验夹具的建设
用于构建实验夹具（ 图4）的机械部件的描述从协议中省略，因为它可以以各种方式来构造。在此示范标准光学机械部件和支撑夹具用于安装头枕杆和身体约束管（参见材料节）。类似光学机械零件可以被用来在电极保持器安装到机动显微。重要的是在夹具的头部约束杆被安装在相同的螺距角所用外科手术期间…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We are grateful to the Canadian Institutes of Health Research (grant MT-5877), the National Institutes of Health (grants NS058668 and NS066664), and the US-Israeli Binational Foundation (grant 2003222) for funding these studies.
Materials
| Name of the Reagent | Company | Catalogue Number | Comments |
| Ketaset (Ketamine HCl) | Fort Dodge | N/A | |
| Anased (Xylazine solution) | Lloyd Laboratories | N/A | |
| Betadyne (Povidone-Iodine) | CVS Pharmacy | 269281 | |
| Loctite 495 | Grainger Industrial Supply | 4KL86 | 20-40 cp cyanoacrylate |
| Vetbond | 3M | 1469SB | |
| Grip cement powder | Dentsply Intl | 675571 | For the base of the recording chamber |
| Grip cement liquid | Dentsply Intl | 675572 | For the base of the recording chamber |
| Silicone Gel | Dow Corning | Mar-80 | |
| Jet denture repair acrylic powder | Lang Dental Manufacturing Co. | N/A | For securing the head restraint apparatus to the cranium |
| Ortho-Jet Fast curing orthodontic acrylic resin liquid | Lang Dental Manufacturing Co. | N/A | For securing the head restraint apparatus to the cranium |
| Chicago sky blue | Sigma | C8679 | |
| Paraformaldehyde | Sigma | 158127 | For perfusion and tissue fixation |
| Phosphate-buffered saline | Sigma | P3813 | For perfusion and tissue fixation |
| Cytochrome C | Sigma | C2506 | For cytochrome-oxidase staining (Figure 7) |
| Diaminobenzidine | Sigma | D5905 | For cytochrome-oxidase staining (Figure 7) |
| Material Name | Company | Catalogue Number | Comments |
| Rat sock | Sew Elegant (San Diego, CA) | N/A | Custom made, Figures 1, 4 |
| PVC tube 2 ½” | U.S. Plastic Co. | 34108 | Figure 4 |
| Subminiature D pins & sockets | TE Connectivity | 205089-1 | Figure 3 |
| Stainless steel music wire 0.010” diameter | Precision Brand Products, Inc. | 21010 | Figure 3 |
| Stereotaxic holding frame | Kopf Instruments | Model 900 | Figure 3 |
| Stereotaxic ear bars | Kopf Instruments | Model 957 | Figure 3 |
| Stereotaxic manipulator | Kopf Instruments | Model 960 | Figure 3 |
| ½ mm drill burr | Henry Schein | 100-3995 | |
| Quiet-Air dental drill | Midwest Dental | 393-1600 | |
| Stainless steel 0-80 1/8” screw | Fastener superstore | 247438 | Figure 3 |
| 0.2mL centrifuge tube | Fisher Scientific | 05-407-8A | Figure 3 |
| Custom head-holding bar | UCSD SIO Machine Shop | N/A | Custom made, Figures 2, 3, 4 |
| Custom head-holding plate | UCSD SIO Machine Shop | N/A | Custom made, Figure 2, 3, 4 |
| Right angle post-clamp | Newport | MCA-1 | Figure 3,4; standard opto-mechanical parts for the experimental jig (Figure 4) are also from Newport Corp. |
| 8-32 3/4” screw | Fastener Superstore | 240181 | For head-restraint, Figure 3 |
| 4-40 ¼” screw | Fastener Superstore | 239958 | For head restraint, Figures 3, 4 |
| Quartz capillary tubing | Sutter Instruments | QF-100-60-10 | Figure 5 |
| Carbon dioxide laser puller | Sutter instruments | P-2000 | |
| Motorized micromanipulator | Sutter Instruments | MP-285 | |
| Microelectrode amplifier | Molecular Devices | Multiclamp 700B | Alternate part: Molecular Devices Axoclamp 900A |
| Microelectrode amplifier head stage | Molecular Devices | CV-7B | Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A |
| Isolated pulse stimulator | A-M Systems | Model 2100 | Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A |
| Audio monitor | Radio Shack | 32-2040 | |
| Pipette holder | Warner Instruments | #MEW-F10T | Alternate parts: see Discussion |
| Figure 6A | |||
| Electrode lead wire | Cooner wire | NEF34-1646 | (optional), Figure 6D |
| Relay for amplifier head-stage | COTO Technology | #2342-05-000 | (optional) Used with a custom-made printed circuit board (UCSD Physics Electronics Shop), Figure 6A-C |
| Digital video camera | Basler | A602fm | (optional) For behavioral monitoring, Figure 7 |
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